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Autophagy dysfunction is a hallmark of type 1 diabetes. However, the precise molecular mechanism of proteinuria-induced dysfunctional autophagy remains unclear. Herein, we investigated the role of programmed cell death 4 (PDCD4) in the regulation of autophagy in the pathogenesis of diabetic kidney disease (DKD) in vivo and in vitro. RT-qPCR, immunohistochemistry (IHC), and western blotting demonstrated an upregulation of Pdcd4 mRNA and protein in streptozotocin (STZ)-induced DKD rats, as compared to the control. In addition, IHC and western blotting of a unilateral ureteral obstruction mouse model showed an upregulation of PDCD4 in the disease group, as compared to their respective controls. IHC analysis of kidney biopsy samples of human DKD patients showed an upregulation of PDCD4 compared to the control. Western blotting of the STZ-induced DKD rat tissues displayed a low microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, as compared to the control. It was found that albumin overload in cultured PTECs upregulated the expression of PDCD4 and p62 and decreased the expression of LC3-II and autophagy-related 5 (Atg5) proteins. The knockout of Pdcd4 in cultured PTECs could reduce albumin-induced dysfunctional autophagy, as evidenced by the recovery of Atg5 and LC3-II protein. The forced expression of PDCD4 could further suppress the expression of the crucial autophagy-related gene Atg5. Evidence suggests that endogenous PDCD4 promotes proteinuria-induced dysfunctional autophagy by negatively regulating Atg5. Therefore, PDCD4 may be a potential therapeutic target in DKD. Key words: autophagy, programmed cell death 4, diabetes kidney disease, Atg5. La dysfonction de l'autophagie est une caractéristique du diabète de type 1. Toutefois, le mécanisme moléculaire précis de la dysfonction de l'autophagie induite par la protéinurie n'est pas compris. Dans cette étude, le rôle de PDCD4 (<< programmed cell death 4 >>) dans la régulation de l'autophagie dans la pathogenèse de la maladie rénale diabétique (MRD) in vivo et in vitro est décrit. La RT-qPCR, l'immunohistochimie (IHC) et le buvardage Western ont démontré une régulation à la hausse de l'ARNm et de la protéine PDCD4 chez les rats avec MRD induite par la streptozotocine (STZ), par rapport au contrôle. En outre, l'IHC et le buvardage Western dans un modèle d'obstruction urétérale unilatérale chez la souris ont montré une régulation à la hausse de PDCD4 dans le groupe malade, par rapport à leur contrôle respectif. L'analyse en IHC d'échantillons de biopsies rénales de patients humains atteints de MRD a montré une régulation à la hausse de PDCD4 par rapport au contrôle. Le buvardage western de tissus de rats avec une MRD induite par la STZ a montré une faible présence de LC3-II (<< microtubule-associated protein 1A/1B-light chain 3 >>), par rapport au contrôle. Il a été constaté que la surcharge d'albumine dans les cellules épithéliales de tubules proximaux (PTEC) en culture pouvait réguler à la hausse l'expression de PDCD4 et de p62, et diminuer l'expression de LC3-II et de la protéine liée à l'autophagie Atg5. Le knockout de PDCD4 dans les PTEC en culture pouvait réduire l'autophagie dysfonctionnelle induite par l'albumine, comme le montre la récupération des proteins Atg5 et LC3-II. L'expression forcée de PDCD4 pouvait également supprimer l'expression d'Atg5, un gène crucial lié à l'autophagie. Dans cette étude, les preuves suggèrent que la PDCD4 endogène favorise l'autophagie dysfonctionnelle induite par la protéinurie en régulant négativement Atg5. La PDCD4 pourrait donc être une cible thérapeutique potentielle dans la MRD. [Traduit par la Rédaction] Mots-clés : autophagie, mort cellulaire programmée 4, maladie rénale diabétique, Atg5., Introduction Diabetic kidney disease (DKD) is a leading diabetes complication that develops into a severe health care crisis because of its high incidence and huge financial burden (Andersen et al. [...]

Diabetes mellitus (DM) is a metabolic disorder that causes severe complications. Thus, the aims of this study were to investigate the influence of DM and folic acid treatment on liver and renal biomarkers, and heart remodeling through evaluation of cardiac matrix metalloproteinase (MMP) activity. There were 4 groups: control (physiological saline 1 mL/kg, i.p., 28 days), DM (streptozotocin [STZ] 100 mg/kg in physiological saline, i.p., 1 day), folic acid (FA; 5 mg/kg, i.p., 28 days), and DM+FA (STZ 100 mg/kg, i.p., 1 day and folic acid 5 mg/kg, i.p., 28 days). Our results demonstrated increased aminotransferase and alkaline phosphatase activity, urea and creatinine concentration, and decreased albumin and fibrinogen concentration in the DM group. MMP-2 relative activity was elevated in the DM and FA groups; MMP-9 was decreased in the DM and increased in the FA group. The folic acid treatment of diabetic rats did not change aminotransferase activity; it alleviated the increase in alkaline phosphatase and the decrease in albumin and fibrinogen concentration, and reduced MMP-2 activity; however, it increased urea and creatinine concentration. In conclusion, folic acid treatment of diabetic rats has cardio- and hepato-protective effects. However, its dosing should be carefully considered because of possible renal damage. Key words: diabetes mellitus, cardiac remodelling, matrix metalloproteinases, hepatic damage, renal damage, rat. Le diabete sucre (DS) est une maladie metabolique qui cause des complications graves. Par consequent, ces travaux avaient pour but d'etudier l'influence du DS et de l'administration d'acide folique (AF) sur les biomarqueurs hepatiques et renaux, ainsi que sur le remodelage du creur a l'aide de l'evaluation de l'activite des metalloproteinases matricielles cardiaques. Nous avons forme 4 groupes : temoin (solution physiologique a 1 ml/kg, i.p. pendant 28 jours), DS (streptozotocine [STZ] a 100 mg/kg dans une solution physiologique, i.p. pendant 1 jour), AF (a 5 mg/kg dans une solution physiologique, i.p. pendant 28 jours) et DS + AF (STZ a 100 mg/kg dans une solution physiologique, i.p. pendant 1 jour et AF a 5 mg/kg dans une solution physiologique, i.p. pendant 28 jours). Nos resultats ont montre une hausse de l'activite des aminotransferases et de la phosphatase alcaline, des concentrations d'uree et de creatinine, ainsi qu'un abaissement des concentrations d'albumine et de fibrinogene dans le groupe DS. Nous avons observe une hausse de l'activite relative de la MMP-2 dans les groupes DS et AF, ainsi qu'un abaissement de la MMP-9 dans le groupe DS, mais une hausse dans le groupe AF. L'administration AF n'a pas entraine de modification de l'activite des aminotransferases chez les rats diabetiques : il entrainait une attenuation de la hausse de la phosphatase alcaline, ainsi qu'un abaissement des concentrations d'albumine et de fibrinogene, avec une diminution de l'activite de la MMP-2; il entrainait toutefois une hausse des concentrations d'uree et de creatinine. En conclusion, l'administration AF permet d'obtenir des effets cardio- et hepatoprotecteurs chez des rats diabetiques. Cependant, il faudrait entreprendre une augmentation des doses AF avec prudence en raison de la possibilite d'observer des lesions renales. [Traduit par la Redaction] Mots-cles: diabete sucre, remodelage cardiaque, metalloproteinases matricielles, dommages hepatiques, dommages renaux, rat., Introduction Diabetes mellitus (DM) is a global public health problem with increasing incidence and prevalence (Danaei et al. 2011). It is a group of metabolic disorders characterized by hyperglycemia; altered [...]

Learning and memory processes can be influenced by recent experience, but the mechanisms involved are poorly understood. Enhanced plasticity during critical periods of early life is linked to differentiating parvalbumin (PV)-interneuron networks (1-7), suggesting that recent experience may modulate learning by targeting the differentiation state of PV neurons (8-11) in the adult. Here we show that environmental enrichment and Pavlovian contextual fear conditioning induce opposite, sustained and reversible hippocampal PV-network configurations in adult mice. Specifically, enrichment promotes the emergence of large fractions of low-differentiation (low PV and GAD67 expression) basket cells with low excitatory-to-inhibitory synaptic-density ratios, whereas fear conditioning leads to large fractions of high-differentiation (high PV and GAD67 expression) basket cells with high excitatory-to-inhibitory synaptic-density ratios. Pharmacogenetic inhibition or activation of PV neurons was sufficient to induce such opposite low-PV-network or high-PV-network configurations, respectively. The low-PV-network configuration enhanced structural synaptic plasticity (12,13), and memory consolidation and retrieval, whereas these were reduced by the highPV-network configuration. We then show that maze navigation learning (14) induces a hippocampal low-PV-network configuration paralleled by enhanced memory and structural synaptic plasticity throughout training, followed by a shift to a high-PV-network configuration after learning completion. The shift to a low-PV-network configuration specifically involved increased vasoactive intestinal polypeptide (VIP)-positive GABAergic boutons and synaptic transmission onto PV neurons (15,16). Closely comparable low- and highPV-network configurations involving VIP boutons were specifically induced in primary motor cortex upon rotarod motor learning (17,18). These results uncover a network plasticity mechanism induced after learning through VIP-PV microcircuit modulation (19), and involving large, sustained and reversible shifts in the configuration of PV basket-cell networks in the adult. This novel form of experience-related plasticity in the adult modulates memory consolidation, retrieval and learning, and might be harnessed for therapeutic strategies to promote cognitive enhancement and neuroprotection., To determine whether plasticity and learning in the adult may involve alterations in the differentiation state of PV-positive GABA (γ-aminobutyric acid)-ergic interneurons, we monitored PV immunoreactivity in ensembles of genetically [...]

Carbon 13 nuclear magnetic resonance (NMR) isotopomer analysis was used to measure the rates of oxidation of long-chain fatty acids, ketones, and pyruvate to determine the minimum pyruvate concentration ([pyruvate]) needed to suppress oxidation of these alternative substrates. Substrate mixtures were chosen to represent either the fed or fasted state. At physiological [pyruvate], fatty acids and ketones supplied the overwhelming majority of acetyl-CoA. Under conditions mimicking the fed state, 3 mM pyruvate provided ~80% of acetylCoA, but under fasting conditions 6 mM pyruvate contributed only 33% of acetyl-CoA. Higher [pyruvate], 10-25 mM, was associated with transient reduced cardiac output, but overall hemodynamic performance was unchanged after equilibration. These observations suggested that 3-6 mM pyruvate in the coronary arteries would be an appropriate target for studies with hyperpolarized [1-[sup.13]C]pyruvate. However, the metabolic products of 3 mM hyperpolarized [1-[sup.13]C]pyruvate could not be detected in the isolated heart during perfusion with a physiological mixture of substrates including 3% albumin. In the presence of albumin even at high concentrations of pyruvate, 20 mM, hyperpolarized [H.sup.13]C[O.sup.-.sub.3] could be detected only in the absence of competing substrates. Highly purified albumin (but not albumin from plasma) substantially reduced the longitudinal relaxation time of [1-[sup.13]C]pyruvate. In conclusion, studies of cardiac metabolism using hyperpolarized [1-[sup.13]C]pyruvate are sensitive to the effects of competing substrates on pyruvate oxidation. myocardium; pyruvate; dynamic nuclear polarization; substrate oxidation; isolated rat heart; carbon-13; nuclear magnetic resonance doi: 10.1152/ajpheart.00656.2009.

The aim of this study was to assess the effects of nutrient ingestion, dietary protein intake, age, and sex on the fractional synthesis rate (FSR) of albumin. Thirty-six healthy free-living individuals (8 females and 10 males aged 21-43 y and 9 females and 9 males aged 63-79 y) completed three 18-d periods of controlled feeding with protein intakes of 125% (P125, 1.00 g protein x [kg.sup.-1] x [d.sup.-1]), 94% (P94, 0.75 g protein x [kg.sup.-1] x [d.sup.-1]), and 63% (P63, 0.50 g protein x [kg.sup.-1] x [d.sup.-1]) of the recommended dietary allowance. On d 12 of each trial, postabsorptive (PA) serum albumin concentration was determined and PA and postprandial (PP) albumin FSR were estimated from the rate of L-[1-[sup.13]C] leucine incorporation into plasma albumin during an 8-h infusion. There were no age-related differences in PA and PP albumin FSR. Albumin FSR was higher PP than PA (P < 0.0001), and the increase in albumin FSR from PA to PP was smaller as dietary protein intake decreased from P125 to P94 and P63 (P< 0.05). Independent of protein intake, males had a higher albumin FSR (P< 0.05) and a greater increase in albumin FSR with feeding (P < 0.05). There was no age or dietary protein effect on serum albumin concentrations, but males had higher albumin concentrations than females (P < 0.0001). These results show that older persons are responsive to nutrient ingestion and dietary protein-related changes in albumin FSR. The greater albumin synthesis rate in males might contribute to a higher albumin concentration set point.

In view of the potential involvement of peripherally synthesized, circulating amphipathic mediators [such as platelet-activating factor (PAF) and prostaglandin [E.sub.2]] in the systemic inflammatory response to lipopolysaccharide (LPS), we hypothesized that transport of amphipaths by albumin is essential for conveying peripheral inflammatory signals to the brain. Our first specific aim was to test this hypothesis by studying LPS-induced fever and hypothermia in Nagase analbuminemic rats (NAR). NAR from two different colonies and normalbuminemic Sprague-Dawley rats were preimplanted with jugular catheters, and their febrile responses to a mild dose of LPS (10 [micro]g/kg iv) at thermoneutrality and hypothermic responses to a high dose of LPS (500 [micro]g/kg iv) in the cold were studied. NAR of both colonies developed normal febrile and hypothermic responses, thus suggesting that transport of amphipathic mediators by albumin is not indispensable for LPS signaling. Although alternative carrier proteins [such as [[alpha].sub.1]-acid glycoprotein (AGP)] are known to assume transport functions of albumin in NAR, it is unknown whether inflammatory mediators are capable of inducing their actions when bound to alternative carriers. To test whether PAF, the most potent amphipathic pyrogen, causes fever when administered in an AGP-bound form was our second aim. Sprague-Dawley rats were preimplanted with jugular catheters, and their thermal responses to infusion of a 1:1 [PAF-AGP] complex (40 nmol/kg iv), AGP (40 nmol/kg iv), or various doses of free (aggregated) PAF were studied. The complex, but neither free PAF nor AGP, caused a high (~1.5[degrees]C) fever with a short ( systemic inflammation; fever; hypothermia; body temperature; platelet-activating factor; Nagase analbuminemic rats

Normal albumin loss from the plasma is thought to be minimized by a number of mechanisms, including charge repulsion with the capillary wall and an intracellular rescue pathway involving the major histocompatibility complex-related Fc receptor (FcRn)-mediated mechanism. This study investigates how these factors may influence the mechanism of hypoalbuminemia. Hypoalbuminemia in rats was induced by treatment with puromycin aminonucleoside (PA). To test the effects of PA on capillary wall permeability, plasma elimination rates were determined for tritium-labeled tracers of different-sized Ficolls, negatively charged Ficolls, and [sup.14]C-labeled tracer of albumin in control and PA-treated Sprague-Dawley rats. Urinary excretion and tissue uptake were also measured. Hypoalbuminemia was also examined in two strains of FcRn-deficient mice: [[beta].sub.2]-microglobulin ([[beta].sub.2]M) knockout (KO) mice and FcRn [alpha]-chain KO mice. The excretion rates of albumin and albumin-derived fragments were measured. PA-induced hypoalbuminemia was associated with a 2.5-fold increase in the plasma elimination rate of albumin. This increase could be completely accounted for by the increase in urinary albumin excretion. Changes in the permeability of the capillary wall were not apparent, inasmuch as there was no comparable increase in the plasma elimination rate of 36- to 85-[Angstrom] Ficoll or negatively charged 50- to 80-[Angstrom] Ficoll. In contrast, hypoalbuminemic states in [[beta].sub.2]M and FcRn KO mice were associated with decreases in excretion of albumin and albumin-derived fragments. This demonstrates that the mechanism of hypoalbuminemia consists of at least two distinct forms: one specifically associated with the renal handling of albumin and the other mediated by systemic processes. albumin; capillary wall permeability; glomerular permeability; macromolecular transport probes; plasma elimination rate: [[beta].sub.2]-microglobulin; Fc receptor

Our aim was to examine the effect of acute inflammation localized in the colon and early macronutrient restriction on protein synthesis in a piglet model. In a 2 x 2 factorial design, piglets (n = 32) were fed an adequate or macronutrient-restricted diet with or without dextran sulfate-induced colitis for 7 d. The stable isotope tracer L-[5,5,[5.sup.-2][H.sub.3]]leucine was infused to determine protein kinetics at the whole-body level and synthesis of tissue and plasma proteins. In the well-nourished state, colitis did not affect weight gain or protein kinetics except for an increase in albumin synthesis (P < 0.05). Macronutrient restriction alone caused a general slowing of protein metabolism including decreased weight gain (P < 0.0004), whole-body protein turnover (P < 0.0001), and liver (P < 0.01) and plasma protein (P < 0.03) synthesis. However, in the presence of macronutrient restriction, colitis compromised weight gain further (P < 0.02) and decreased muscle protein synthesis (P < 0.05) due to a redistribution of protein metabolism that supported enhanced synthesis of plasma proteins. The increased contribution of plasma protein synthesis to whole-body protein turnover was attributable mainly to increased synthesis of albumin (P < 0.006). Concentrations of plasma proteins were unaffected despite dramatic changes in their synthesis rates, thereby underestimating the effects of malnutrition and colitis on protein metabolism. Increased synthesis of plasma proteins, particularly the negative acute phase reactant albumin, compromises weight gain and muscle protein synthesis only when macronutrient intake is inadequate, underscoring the role of adequate nutrition in preventing growth impairment and muscle wasting in acute inflammation. These results suggest that the hypoalbuminemia of inflammatory bowel disease should not be attributed to decreased synthesis. J. Nutr. 133: 1875-1881, 2003. KEY WORDS: * piglets, protein synthesis * inflammation, albumin * malnutrition

The mechanisms leading to hypoalbuminemia in sepsis were explored by measuring plasma volume, albumin distribution, plasma albumin transcapillary escape rate (TER), and efflux (TER x albumin intravascular pool). These parameters were quantified in infected rats, injected intravenously with live Escherichia coli, and pair-fed and well-fed rats using an injection of [sup.35]S-albumin and measuring plasma and whole body albumin concentrations. Animals were studied on days 1, 6, and 10 after infection. In pair-fed rats, neither albumin distribution nor exchange rate between the intra- and extravascular compartments was modified. The increase of plasma volume after infection partly explained hypoalbuminemia. Infection resulted in a reduction of the total albumin pool of the body all along the experimental period, indicating a net loss of the protein. Albumin TER (%/day) was significantly increased 1 and 6 days after infection, but the absolute efflux was increased only on day 1. Normal values were observed on day 10. Therefore, an accelerated plasma efflux contributes to hypoalbuminemia only during the early period of sepsis. During this phase, the protein was retained in the extravascular space where it was probably catabolized. Later on, other factors are probably involved. plasma albumin escape; albumin distribution; infection

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral (ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical (ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of ~1.6 [micro]M and maximal transport rate of ~0.15 fmol*[cm.sup.-2]*[s.sup.-1]. At 15 and 4[degrees]C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an ~60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung. protein transport; air-blood barrier; saturable transcytosis; albumin-binding sites; alveolar fluid balance

Alveolar lining fluid normally contains proteins of important physiological, antioxidant, and mucosal defense functions [such as albumin, immunoglobulin G (IgG), secretory IgA, transferrin, and ceruloplasmin]. Because concentrations of plasma proteins in alveolar fluid can increase in injured lungs (such as with permeability edema and inflammation), understanding how alveolar epithelium handles protein transport is needed to develop therapeutic measures to restore alveolar homeostasis. This review provides an update on recent findings on protein transport across the alveolar epithelial barrier. The use of primary cultured rat alveolar epithelial cell monolayers (that exhibit phenotypic and morphological traits of in vivo alveolar epithelial type I cells) has shown that albumin and IgG are absorbed via saturable processes at rates greater than those predicted by passive diffusional mechanisms. In contrast, secretory component, the extracellular portion of the polymeric immunoglobulin receptor, is secreted into alveolar fluid. Transcytosis involving caveolae and clathrincoated pits is likely the main route of alveolar epithelial protein transport, although relative contributions of these internalization steps to overall protein handling of alveolar epithelium remain to be determined. The specific pathways and regulatory mechanisms responsible for translocation of proteins across lung alveolar epithelium and regulation of the cognate receptors (e.g., 60-kDa albumin binding protein and IgG binding FcRn) expressed in alveolar epithelium need to be elucidated. alveolar epithelial cells; albumin; secretory component; immunoglobulin G

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of [sup.125]I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol * [min.sup.-1] * [10.sup.6] [cells.sup.-1]. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-[beta]-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased [sup.125]I-albumin uptake 2.3-fold. At 37[degrees]C, [sup.125]I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (I[C.sub.50] = 1 [micro]M). Using a two-site model, we estimated by Scatchard analysis the affinity ([K.sub.D]) and maximal capacity ([B.sub.max]) of albumin uptake to be 0.87 [micro]M ([K.sub.D1]) and 0.47 pmol/[10.sup.6] cells ([B.sub.max1]) and 93.3 [micro]M ([K.sub.D2]) and 20.2 pmol/[10.sup.6] cells ([B.sub.max2]). At 4[degrees]C, we also observed two populations of specific binding sites, with high ([K.sub.D1] = 13.5 nM, 1% of the total) and low ([K.sub.D2] = 1.6 [micro]M) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer. capillary permeability; vesicular transport; caveolae; albumin-binding glycoprotein gp60

In children, aortic lipid deposition develops in triangular regions of the wall downstream of branch points, whilst in adults these regions are particularly free of disease. Comparable age-related patterns occur in rabbit aortas. They may be explained by patterns of wall permeability to circulating macromolecules: along the longitudinal midline through branches, permeability is greater downstream than upstream in immature rabbits, but is greater upstream at later ages. Here we have mapped permeability in detail around such branches, not just along the midline. Short-term uptake of rhodamine-labeled albumin, measured using digital imaging fluorescence microscopy of serial sections, was greatest in an approximately triangular region downstream of immature branches, but in mature animals it was greater upstream, particularly away from the midline, and in streaks to the side of branches. Hence the maps are consistent with earlier permeability data and closely resemble the patterns of disease. [DOI: 10.1115/1.1517063]

Because modification of plasma albumin on tyrosine residues generates nitrated albumin (NOA) that may function as a mechanism of nitrogen monoxide clearance from microcirculation, we investigated biochemicaly and morphologically the cell surface binding and the transendothelial transport of NOA. An electron microscopic study was carried out with mouse lungs and hearts perfused in situ with NOA and NOA-Au complexes. The results indicate that NOA-Au can bind to the endothelial cell surface, and its binding can be blocked by albumin plus nitrotyrosine (NO-tyrosine) or abolished by excess NOA. We detected NOA-Au into perivascular spaces as early as 30 sec after the beginning of its perfusion. NOA, unlike native albumin , leaves the vascular lumina via both endothelial caveolae and open junctions. By cross-linking and ligand blotting analysis, we showed that NOA interacted with the same albumin binding proteins of 16-18, 30-32, 60, and 74 kDa as native albumin. ELISA performed on tissue homogenates obtained from the same specimens showed that NOA transport was 2- to 4-fold greater than native albumin. The augmented transendothelial transport of NOA reflects its transcytosis as well as its exit from the microcirculation via open junctions. The increased transport of NOA may serve as an important mechanism that protects a vascular bed against the damaging effects of nitrogen monoxide and peroxynitrite.

The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but [sup.125]I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 [micro] M) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin. endocytic receptors; endocytosis

Nitric oxide (N[O.sup.*]) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological N[O.sup.*] concentrations, plasma albumin becomes saturated with N[O.sup.*] and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of N[O.sup.*] oxidation in the albumin hydrophobic core and specific transfer of N[O.sup.+] to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of N[O.sub.*] and its reactive oxides and controls the dynamics of N[O.sup.*]-dependant processes in the vasculature.

Impairment of albumin and whole body postprandial protein synthesis in compensated liver cirrhosis. Am J Physiol Endocrinol Metab 282: E304-E311, 2002; 10.1152/ajpendo. 00333.2001.--To investigate the anabolic effects of feeding in cirrhosis, we measured albumin fractional synthesis rate (FSR) and whole body protein synthesis in six nondiabetic patients with stable liver cirrhosis (three in the Child-Pugh classification Class A, three in Class B) and in seven normal control subjects, before and after administration of a 4-h mixed meal. Leucine tracer precursor-product relationships and whole body kinetics were employed at steady state. Basal levels of postabsorptive albumin concentration and FSR, whole body leucine rate of appearance, oxidation, and nonoxidative leucine disposal (NOLD, [approximately equal to] protein synthesis) were similar in the two groups. However, after the meal, in the patients neither albumin FSR (from 8.5 [+ or -] 1.5 to 8.8 [+ or -] 1.8 %/day) nor NOLD (from 1.69 [+ or -] 0.22 to 1.55 [+ or -] 0.26 [micro]mol * [kg.sup.-1] * [min.sup.-1]) changed (P = nonsignificant vs. basal), whereas they increased in control subjects (albumin FSR: from 10.9 [+ or -] 1.5 to 15.9 [+ or -] 1.9 %/day, P < 0.002; NOLD: from 1.80 [+ or -] 0.14 to 2.10 [+ or -] 0.19 [micro]mol * [kg.sup.-1] * [min.sup.-1], P = 0.032). Thus mixed meal ingestion did not stimulate either albumin FSR or whole body protein synthesis in compensated liver cirrhosis. The mechanism(s) maintaining normoalbuminemia at this disease stage need to be further investigated. albumin synthesis; nonoxidative leucine disposal; meal ingestion; [alpha]-ketoisocaproate; specific activity; compensated cirrhosis

Research has been conducted on the gadolinium(III) complexes. Results indicate that the water relaxivity of these complexes formed with the fatty acid analogues of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonic acid) increase in the presence of albumin.

Vogel, Stephen M., Richard D. Minshall, Milena Pilipovic, Chinnaswamy Tiruppathi, and Asrar B. Malik. Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein. Am J Physiol Lung Cell Mol Physiol 281: L1512-L1522, 2001.--The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall [sup.125.I]-albumin permeability-surface area (PS) product 2.5-fold without affecting the capillary filtration coefficient ([K.sub.f,c]; a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both [K.sub.f,c] and [sup.125.I]-albumin PS product. Increasing perfusate albumin concentration to > 1 g/100 ml (E[C.sub.50] 1.2 g/100 ml) was sufficient to block [sup.125.I]-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in [sup.125.I]-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a nonhydraulic pathway that fits with a model of albumin permeability via the transcellular pathway. rat lung; filipin; gp60; antibody cross-linking

Wang, Peiyi, Alexander D. Verin, Anna Birukova, Lydia I. Gilbert-McClain, Keri Jacobs, and Joe G. N. Garcia. Mechanisms of sodium fluoride-induced endothelial cell barrier dysfunction: role of MLC phosphorylation. Am J Physiol Lung Cell Mol Physiol 281: L1472-L1483, 2001.-NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicatlng endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of [Ca.sup.2+]/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction. Rho-associated kinase; myosin-specific phosphatase; transendothelial electrical resistance; actin cytoskeletal rearrangement; myosin light chain

Minnear, Fred L., Sandeep Patil, Donald Bell, Jonathan P. Gainor, and Christine A. Morton. Platelet lipid(s) bound to albumin increases endothelial electrical resistance: mimicked by LPA. Am J Physiol Lung Cell Mol Physiol 281: L1337-L1344, 2001.--The objectives were to determine whether the permeability-decreasing activity of platelet-conditioned medium (PCM) is associated with a lipid bound to albumin and whether lysophosphatidic acid (LPA) is present in the PCM. A decrease in permeability was assessed by an increase in electrical resistance across endothelial cell monolayers derived from bovine pulmonary arteries and microvessels. The Sephacryl S-200 fraction of PCM that contained albumin, the albumin immunoprecipitate from the PCM, and the methanol extract from the albumin immuno-precipitate all increased endothelial electrical resistance. Increased electrical resistance induced by PCM was not abolished by boiling and was mimicked by 1-oleoyl-LPA and l-palmitoyl-LPA. Analysis of a methanol-chloroform extract of one sample of PCM by electrospray mass spectrometry revealed many fatty acids, ceramide, diacylglycerol, phosphatidic acid, and palmitoyl-LPA, but analysis of a second sample of PCM and the methanol extract of its albumin immu-noprecipitate revealed no LPA, only lipids. These findings indicate that a bioactive lipid(s), possibly LPA, released from platelets and subsequently bound to albumin forms an active complex that decreases endothelial permeability. lysophosphatidic acid; gel electrophoresis; immunoprecipitation; methanol extraction; gel filtration chromatography; Blue Sepharose chromatography; electrospray mass spectromery Received 8 December 2000; accepted in final form 20 July 2001

This article compares the effects of active recovery and rest recovery on concentrations of free fatty acids in trained males after running. Findings indicate that active recovery, or fifteen minutes of light running, resulted in lower peaks and overall concentrations of free fatty acids when compared to complete rest recovery.

Byline: X. L. Qiu (1), L. V. Brown (1), S. Parameswaran (1), V. W. Marek (2), G. S. Ibbott (3), S. J. Lai-Fook (1) Keywords: Key words: Lung interstitium--Albumin diffusion--Hyaluronidase--Rabbit. Abstract: Albumin diffusion measured in an isolated segment of rabbit lung interstitium with a radioactive tracer [(.sup.125]I-albumin) technique was independent of albumin concentration and similar to the free diffusion of albumin in water (Qiu et al, 1998. J Appl Physiol 85: 575--583). We studied the effect of hyaluronidase on the diffusion of albumin. Isolated rabbit lungs were inflated with silicon rubber by way of airways and blood vessels, and two chambers were bonded to the sides of a [proportional to]0.5-cm thick slab enclosing a vessel with an interstitial cuff. One chamber was filled with 2 g/dl albumin solution containing 125.sup.I-albumin and 0.02 g/dl hyaluronidase. Unbound 125.sup.I was removed from the tracer by dialysis before use. The other chamber filled with Ringer&apos;s solution was placed within a NaI(Tl) scintillation detector. Diffusion of tracer was measured continuously for 120 h. Albumin diffusion coefficient (D) and interstitial area (A) were obtained by fitting the tracer-time curve with the theoretical solution of the equation describing one-dimension diffusion of a solute across a membrane. D averaged 5.2 x 10.sup.-7 cm.sup.2/s for albumin diffusion with hyaluronidase, 20% less than that measured previously without hyaluronidase. Hyaluronidase had no effect on A. Results indicated an interaction between albumin and interstitial hyaluronan that was the opposite of the steric effect on albumin excluded volume measured in solution. Author Affiliation: (1) Center for Biomedical Engineering, University of Kentucky, Lexington, Kentucky, 40506, US (2) Department of Computer Science, University of Kentucky, Lexington, Kentucky, 40506, US (3) Department of Radiation Medicine, University of Kentucky, Lexington, Kentucky, 40506, US Article note: Accepted for publication: 9 May 1999

The effects of mannitol and albumin in combination with other cardioplegia enhancers during 24-h cold heart preservation and warm reperfusion in the isolated rat heart were investigated. It was found that the cardiac function of mannitol-treated hearts is lower than that of hearts not treated with mannitol with regards to diastolic pressure, relaxation, contractility, coronary flow, myocardial creatine kinase release and lipid peroxidation. Results also show that the performance of albumin-treated hearts was higher than hearts without albumin treatment or mannitol-treated hearts.

A study was conducted to analyze whether glycated albumin modulates protein kinase C activity and if protein kinase C participates in mediating the glycated albumin-induced stimulation of matrix production. The effects of protein kinase c inhibitors on collagen type IV production was examined. Experimental results indicated that Amadori-modified albumin stimulates mesangial cell protein kinase c activity.

The role of fluorescent dyes used in labeling albumin on the inhibition of nitric oxide synthase (NOS) on albumin leakage has been studied. Albumin leakage was observed in rat mesenteric venules during controlled conditions and after exposure to the NOS inhibitor N(super G)-nitro-L-arginine methyl ester (L-NAME). The fluorescent dyes used were FITC, sulforhodamine 101, dichlorotriazinyl aminofluorescein and Oregon Green 514. Results indicated that albumin leakage responses due to L-NAME varied among the four dyes. This finding suggests that caution should be used when comparing albumin leakage results from various dyes.